Abstract (inglese)

Summary

Fragile sites are non-random chromosomal regions prone to breakage, which are expressed as decondensations, gaps or breaks under conditions in which DNA synthesis is partially inhibited. It has been hypothesised that they could represent uncondensed regions of the chromosome, for example due to unreplicated or single-stranded DNA. Moreover, there is some evidences that these loci are involved in chromosomal rearrangements related to tumours (Arlt et al., 2003).
Depending on the frequency of expression in the population and on their inheritance pattern, these regions can be classified in two main categories: rare and common fragile sites.
Rare fragile sites occur in less than 5% of the human population and they are characterized by the presence of di- o trinucleotide repeats (CGG(n) e AT (n)). The rare fragile site expression, segregating as mendelian traits, can be associated with pathological phenotypes. In most of the cases breakages at these sites are due to nucleotide repeat expansions, as occur in the fragile X syndrome. The major group of rare fragile sites is folate-sensitive in vitro, a vitamin involved in the nucleotides biosynthesis, and it can be hypothesized that alterations on the DNA replication can cause the fragility expression within these regions
Common fragile sites (CFS) are thought to be a normal feature of chromosomes and they show an high structural instability as a consequence of a replication stress. These sequences are seen in all individuals, although their expression vary and it is visible only in a subset of cells. CFS are characterised by the presence of AT-rich sequences and show several features of unstable DNA: high frequency of deletions, translocations, hotspots for sister chromatid exchanges (SCE). Moreover, these sites usually span along very large genomic regions and they contain large genes, presenting long intronic sequences (Glover et al., 2005), as demonstrated for FHIT, WWOX and PARK2 located in FRA3B. FRA16D and FRA6E, respectively. Some of them were found to function as tumour suppressor genes (Schwartz et al., 2005) and this evidence supports the hypothesis that the genomic instability can play a role in cancer development.
It has been demonstrated that common fragile sites are late replicating regions, as reported for FRA3B (Le Beau et al., 1998), and the presence of double strand breaks at these sites can appear as a consequence of stalled or collapsed replication forks.
In this study, analysis were focused on common fragile site FRA6E, one of the most frequently CFS expressed in humans. It is located on chromosome 6 (q26) in a susceptibility region frequently found rearranged in cancer. Many genes are located within FRA6E and some of them presented long intronic sequences, as ARID1B and PARK2. Concerning the last one, a putative role of oncosuppressor gene has been suggested. Previous data obtained in our laboratory indicated that FRA6E is a large genomic region of instability, spanning approximately 9 Mb (Russo et al., 2006) and it can be sub-divided in three sub-regions, depending on the flexibility features: the central sub-region is less fragile than the centromeric one, where ARID1B gene is mapping, and than the telomeric one, where PARK2 gene is located.
The aim of this study was to investigate if FRA6E instability could be related to replication defects at this site. Analysis were performed by using an innovative technique, the molecular combing, which allows the uniform elongation of single DNA molecules on silanised glass surfaces, leading to the defined equivalence of 1 ?m = 2 kb. Combining FISH, with specific probes, and immunodetection of replicating DNA on single molecules, it is possible evaluate the replication pattern at specific regions. The first phase of the project consisted in setting up the molecular combing and other related experimental procedures.
The second part of the project was focused on the study of the replication pattern of FRA6E, compared to other two regions: the common fragile site FRA3B and the HPRT locus, as a control region. Concerning FRA6E, the two more fragile regions were considered, where ARID1B and PARK2 genes are located, whereas in FRA3B, the most expressed common fragile site in humans, the core of fragility was analysed, where FHIT gene is mapping.
In order to evaluate the replication pattern at high resolution we used human primary lymphocytes, isolated from two different donors.
For each region, genomic clones were selected by bioinformatics analysis, to be employed as probes in FISH experiments on combed DNA. We focused the analyses in order to map replication origins, to determine fork density, replication rates and deregulative events (unidirectional forks, fork arrest events and asynchronous forks) .
Data collected showed that the mean fork rates seemed to be similar among the regions. In FRA6E the mean fork rates were higher in ARID1B than in PARK2 region, even if they can not be considered significantly different when compared to published data relative to the whole genome (Conti et al., 2007). Looking at the distribution of fork rate values, in ARID1B we noticed a lower variability than in PARK2, and notably forks have been detected on a 200 kb sub-region.
The frequency of unidirectional events was found to be more homogeneous and higher among FRA6E-PARK2 and FRA3B-FHIT than FRA6E-ARID1B and HPRT. Also active origins were mapped and the inter-origin distances were evaluated, obtaining mean values in agreement with already published data (Conti et al., 2007).
Moreover, in order to better understand in which way stress conditions can influence the replication process into the fragile regions, cells were treated with aphidicolin (APH), an inhibitor of the DNA polymerase, at different doses and times. Initially, we verified that the high concentration APH resulted in accumulation of cells in S phase after 24 h of treatment, whereas the cell cycle progression seemed not significantly modified at the other tested conditions.
Analyses at whole genome level performed by molecular combing indicated a strong delaying effect on the replication process after 2 h of treatment, with a strong decrease of the mean fork speed. We also analysed the replication pattern at FRA3B-FHIT, FRA6E-PARK2 and HPRT loci by FISH on interphase nuclei of APH-treated and control cells. APH treatment was found to be effective in slowing replication process, with different extent with respect to the locus considered.
Single locus analysis on APH treated combed DNA, carried out to evaluate the replication pattern of FRA6E-PARK2 and HPRT locus, showed that the mean fork speed in both regions strongly decreased, if compared to the controls, in agreement with the data obtained at whole genome level. At both loci unidirectional forks were found to increase with respect to the controls. Also in this case the inter-origin distances ware evaluated and they were found to be differently affected from APH treatment in HPRT region than in PARK2. Infact, in HPRT the replication stress induced the activation of replication origins that seemed to be inactive in control condition, correlated to a reduced interorigin distance, whereas in PARK2 region the effect of the APH treatment was a strong inactivation of the replication origins.
In order to study the replication of these regions specifically during the S phase, we performed the Centrifugal Elutriation, to obtain synchronous cell sub-populations without any chemical or drug treatments, which may interfere with the fragile site expression. These experiments were performed by using lymphoblastoid cell lines, because the method cannot be used with primary lymphocytes. The achievement of enriched S-phase cell sub-populations will be useful to extend the analyses to the replication process at common fragile sites during the late S-phase, in order to better understand the response to stress conditions.